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The dataset contains 63 records of Chiroptera species collected in continental Portugal and was published by Rebelo et al (2020)". Twenty-six bat species currently known for continental Portugal are represented including the first record of Myotis alcathoe for the country. Bat samples were collected under the scope of several projects spanning from 2005 to 2018. All bats were captured during mist-netting sessions or using harp-traps at roost exits. A non-lethal 3mm wing punch was collected from several individuals and stored in 96% ethanol from where DNA was extracted. The DNA barcoding of these samples was conducted within the InBIO Barcoding Initiative (IBI), funded by EnvMetaGen and PORBIOTA projects. DNA barcode sequences were deposited in BOLD (Barcode of Life Data System) online database. DNA extractions are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources).